1-s2.0-s0022030265882224-main.pdf

PROGESTERONE

IN BOVINE

REPRODUCTION:

A REVIEW

1

W . R. G O M E S AND R. E. ERB Department of Animal Sciences, Purdue University, Lafayette, Indiana ABSTRACT

Portions of an extensive literature were reviewed to evaluate the status of our knowledge relative to progesterone in the bovine. Studies related to the secretion and metabolism of progesterone have been hampered by the lack of chemical methods sufficiently sensitive and specific to quantitate the hormone in snmll samples. Certain bioassays are extremely sensitive but lack specificity and have undefined limits of error. Spectrophotometric determination of progesterone or progesterone derivatives appears specific but is sensitive to only 0.1-0.5 t~g. Fluorometric, gas chromatographic, and double-isotope derivative methods promise sensitivity to 0.01 /zg with adequate specificity and precision. Progesterone and 20 fl-hydroxy-A~-pregnene-3-one a p p e a r to be the principal progestins in the corpus luteum (CL) of cycling and pregnant cows. Progestins have also been reported in bovine ovaries, adrenals, and blood. The CL appears to be the most important source of progesterone throughout bovine pregnancy. The CL from a previous cycle contains measurable progesterone for two or three days after estrus. Progestin in the CL and ovarian vein blood declines rapidly during proestrus from maximum levels observed 14-16 days post-estrus. I n the pregnant cow, CL progestin levels decline about 40% from mid-cycle levels by 42-56 days of pregnancy, further decline slowly until 200 days, then rise again to the 28-day level; after 250 days, a decline to parturition begins. Similar changes are seen in blood levels of the hormones during pregnancy. Progesterone appears important in pregnancy nmintenance, expression of estrus, nornml cyclic function, and in hypophyseal-gonad interrelationships. Thirty years have elapsed since the first isolation of progester(me as a crystalline compound. Since that time, most attempts to use the hormone therapeutically to correct faulty reproductio~ ia the bovine have generally proven unsuccessful. Basic mechanisms involved in the different phases of reproduction are only generally understood, and many details are ahnost completely unknown. Specific sites or qualitative and quantitative aspects of progesterone action and the precise role of the hormone in the hypothalamo-hypophyseal-ovarJan axis (5, 60, 64, 127) remain obscure. F o r many years progesterone (A4-pregnene3,20-dione) was thought to be the only naturally occurring progestational agent. I n more recent years, however, several other compounds have been isolated. The most important of these, quantitatively, appear to be 20 a-hydroxy-A~pregnene-3-one (20 a-ot), 20 fl-hydroxy-A4pregnene-3-oue (20 fl-ol) and 17 a-hydroxy-A~pregnene-3, 20-dione (17-hydroxyprogesterone). I n 1936, the A.I~I.A. Council on Pharmacy and Chemistry (3) adopted the term progestin to indicate progestational agents without regard to the state of chemical purity. Progestin will ~Journal Paper No. 2442, Purdue ~Jniversity Agricultural Experiment Station.

be used in this p a p e r to refer to this group of hormones in biological media or in pure fornL The purpose of this paper is to review the methodology in progestin research and the levels and importance of progestin in the bovine, using comparative data where they appear applicable. No hormonal action is completely independent of the effects of other hormones, and progesterone is no exception; nonetheless, the effects of progestin on reproduction will be isolated here, insofar as is possible. F o r a more complete presentation of hormonal interrelationships, the reader is referred elsewhere (5, 10, 38, 40, 41, 114, 140, 157). BIOLOGICAL A C T I O N S

OF P R O G E S T E R O N E

The physiological and pharmacological actions of progestins have been recently listed (98); therefore, no attempt will be made here to present an all-inclusive discussion of the properties. A general listing of these actions, however, should be valuable for a better understanding of the complexity of the hormonal aspects of reproduction. F i r s t of all, progestins are essential for the uterine development necessary for implantation~ blastocyst development, and maintenance of the fetus and of uterine tone during pregnancy. 314

PROGESTERONE

IN BOVINE

Moreover, progestin may be important as a conditioning agent for normal bovine estrus (92) and may have an effect on tubal and uterine transport of sperm and ova (98). These hormones may inhibit fertilization at certain dose levels in vivo (98) and in vitro (27). Profound effects are also noted on vaginal epithelium (108, 157), ovarian function (31, 49, 106, 142), gonadotrophin elaboration (45, 84), and oxytocin (50) and relaxin (98, 157) actions. Pharmacological doses may antagonize the actions of estrogens or corticoids (32, 98, 157). Many of the above actions are mimicked to one degree or another by progestins other than progesterone, but no compound has yet been found which duplicates exactly :all of the effects of the parent hormone, progesterone. The biological significance of the 20-hydroxy compounds, for example, is not understood; they may well be naturally occurring metabolites of progesterone. Both isomers have twice the activity of progesterone in the Hooker-Forbes intrauterine assay in mice (155). I n the Clauberg rabbit bioassay, 20 fl-ol has 10-20% of the activity of progesterone (80, 155), and 20 a-ol is 30-50% as active (155). A f t e r studying its effects on the endometrium, delay of menstruation, production of withdrawal bleeding, cessation of dysfunctional bleeding, cervical mucus, vaginal smear, and several other factors in the human, Lauritzen (80) reported that 20 fl-ol is a true progestational hormone with nearly half the activity of progesterone. Conversely, 20 fl-ol did not support deciduomata formation in the ovariectomized r a t when 3-mg injections were used (0.5 mg progesterone is effective) (147). I n addition, both 20-hydroxylated compounds failed to maintain pregnancy in the ovariectomized mouse, even with large doses (146). I t seems likely that the biological effectiveness of progestins other than progesterone will renlain obscure until considerably more is known about the mechanisnl of action of these hormones (25, 53, 79, 106, 141). I t is disappointing to note that few results have been forthcoming in this area, although rapid advances are being made toward the elucidation of the p r i m a r y actions of other steroid classes (59, 74, 104, 141). I t is probable that the advent of better methodology will facilitate such studies with the progestins. ASSAY METHODS FOR PROGESTERONE Studies related to the secretion and metabolism of progesterone have been hampered to date by the lack of accurate chemical tech-

315

REPRODUCTION

niques sufficiently sensitive and specific to facilitate the quantitation of the hormone in small plasma samples. Until quite recently it was necessary to collect reproductive tissues at surgery (55, 89) or slaughter (42, 158-161) for the estimation of progesterone. Present methods require the use of large volumes of blood (118). Certain biological assays are extremely sensitive, but lack specificity and have undefined limits of error (36, 98). B I O A S S A ¥ OF P R O G E S T I l q S

Several excellent reviews of progestin bioassay methods are available (98, 108, 157). I n general, the assays based on end points such as complete progestational proliferation, uterine granulosa/nlucosa areas, carbonic anhydrase activity, deciduonlata formation, pregnancy maintenance, or parturition delay [for specific assays and references, see (98, 157)] lack sensitivity and are often difficult and time-consuming. The local progestational tests by McGinty and Hooker and Forbes (see 108, 157) are the most sensitive progesterone assays known, but these tests utilize a subjective end point and lack specificity. Furthermore, these methods have consistently indicated progesterone levels in biological fluids that cannot be verified by chemical assay (36). I n fairness to the bioassay, it should be pointed out that animal responses are essential for the assessment of activity of crude extracts and of individual compounds and are valuable in the isolation of unknown substances. The bioassay has played a vital role in progestin research to date and will be equally important in mechanism of action studies and in the evaluation of the newer synthetic progestins (45, 61, 62). CHEMICAL

~IETI~ODS

Chemical methodology applicable to progesterone research has been reviewed in recent years (101, 114, 154). However, an abundance of new methods has appeared which may be of special significance, depending on the objectives of various research programs dealing with large animal production. Extraction and purification. Extraction of steroids from tissues and fluids has generally been accomplished by the use of organic s01vents or mixtures of solvents (54, 154). Zander (154) has shown that pretreatment of plasma samples with sodium hydroxide as proposed by Short (118) improves the precision of the final progesterone quantitation. I n addition, NaOH decreases the formation of solvent-plasma emulsions during ether extraction.

316

w.R.

G O M E S AND R. E. E R B

One of the difficulties involved in solvent extraction is the great quantity of lipid extracted. Several methods have been utilized to remove this f a t t y material, including centrifugation or filtration in the cold (57, 134) or solvent partitioning (39, 118). A further suggestion has been the use of an acetylation step following extraction. I n this procedure (16), the crude extract is subjected to acetylation following preliminary chromatography. Since progesterone cannot be acetylated, the other impurities may be converted to derivatives which will not remain with progesterone during subsequent chromatography. Zander (154) has reviewed the use of Girard's Reagent T for the separation of ketones from crude extracts, but points out that this procedure has not been applied to routine microanalytical assays for progesterone. To obtain a more nearly pure extract from biological samples, Zaffaroni et al. (153) applied dialysis for the extraction of steroids from blood, but recovery of added progesterone was too low for this method to be valuable. DeVenuto et al. (28) modified the dialysis procedure by extracting continuously in a Soxhlet extractor. Referring to this procedure as perextraction, these workers rePorted 100% recovery of progesterone-C ~ added to the blood. Furthermore, no discernible lipid, protein, or pigment was noted in the extract. The method is limited by the relatively small volume of fluid that can be extracted, but the lack of lipids or chromagens in the extract might further the use of perextraction as quantitative • measurement of progestins becomes nmre sensitive. Separation of individual steroids. Countercurrent distribution has been used for the separation and purification of steroids (83, 100, 101, 108, 114), but has been utilized in only one routine method (83). Application of this excellent method appears to have been limited by the relatively long time required to separate and p u r i f y a single sample. Wilson et al. (149) devised a method usingcolumn chromatography for the separation of steroids on synthetic aluminum silicate with aqueous ethanol. Other workers have used similar systems with silica gel, eelite, aluminum oxide (105, 130), or other supporting media in the columns. Column chromatography is advantageous for the removal of large amounts of impurities, but packing of columns and analysis of individual fractions is very timeconsuming unless automation is applied (4, 48).

The widespread use of p a p e r chromatographic techniques in the separation and identification of progestins is evidence of the advantage of this procedure (15, 16, 102). Since the early use of these systems for the separation of adrenal steroids (15, 16), p a p e r chromatography systems have been devised and studied for the separation of virtually all steroids. Extensive reviews and books on the p a p e r chromatographic separation of steroids are available (16, 102). I n substance, steroid chromatographic systems utilize biphase organic solvents for the separation of compounds on a p a p e r strip or sheet. Resolution, sharp separation, and simplicity are distinct advantages of this tool. A new method of adsorption and partition chromatography--an open column--has been developed in recent years. Thin-layer chromatography (TLC), though in its infancy in steroid research, has already been heralded as a micro-chemical technique that will supplement or even replace p a p e r chromatographic methods. The speed (20-40 min) compared to that of p a p e r chromatography (4-24 hr), the wide range of distinct resolution (5-500 ttg), and the noticeably sharp separation of compounds are distinct advantages of TLC (63). Furthermore, the organic layer on a glass support allows the use of corrosive spray reagents and can be prepared with luminous indicators (115). The method has already been tested for a great number of steroids in various solvent systems and complete separation data are available for progestins (81). Enzymatic methods have become more valua b l e in recent years for the separation and identification of steroids, althogh this has not been their primary role in progestin research. Henning and Zander (67) probably offered the best example of this application of enzymatic methods when they described the separation of 20 a-ol and 20 fl-ol by 20 fl-hydroxysteroid dehydrogenase. The specific enzyme quantitatively converts 20 fl-ol to progesterone but leaves 20 a-ol unchanged. The separation can then be accomplished by chromatography, and the 20 fl-ol can be regenerated by the same enzyme and different cofaetors (67). Quantitation of progesterone. Zarrow et al. (156) confirmed that the ±'-ene-3-one grouping on the steroid nucleus must be present for progestational activity. This same group may be quantitated by its strong absorbance at 240 mg in alcoholic solutions (16, 34). Although the A4-ene-3-one grouping is not specific for progestins, ultraviolet absorption can be a reliable method for their detection and quantitation if suitable separation and purification is ac-

PROGESTERONE

317

IN BOVINE REPRODUCTION

complished. Wiest (145) measured the absorption of progesterone at 240 mt~ by direct spectrophotometric scanning of the paper chromatogram. Other spectrophotometric assays have been reviewed by Zander (154) and are based on the absorbance of ultraviolet light at wave lengths other than 240 m~ by progesterone derivatives. These include the progesterone bisthiosemiearbazone at 300 mtL, the progesterone bisdinitrophenylhydrazone at 380 m~, the bisisonicotinic acid hydrazone at 380 mtL, and the sulfuric acid-ethanol chromagen at 290 mtL. As pointed out by Zander (154), these methods have the advantage of shifting the quantitation wave length away from that of interfering materials, but lack specificity and nmke it difficult to identify the isolated material as progesterone. The limit of sensitivity of the spectrophotometric methods ranges from 0.5 to 1.0 t~g if micro-cells are used in the spectrophotometer. I f not, five times as much hormone is needed (Table 1). Recently, Sommerville et al. (130) reported a modification of the thiosemicarbazide reaction and reported sensitivity to 0.1 t~g using nficro-cells. This is the most sensitive spectrophotometric method reported for progesterone to date, but the specificity problems mentioned above still apply. The fact that other (nonspectrophotometric) assays yield lower results for progesterone levels in nonpregnant women (130, 150, 152) suggests that this method may measure thiosemicarbazone derivatives of other compounds in addition to progesterone, since the progesterone derivative is not isolated prior to spectrophotometric evaluation (130). Fluorometric procedures have been used since 1948 for the quantitation of steroid estrogens (71); however, progesterone exhibits less than 1% of the fluorescence shown by estradiol in sulfuric acid (2, 143). For this reason, this

procedure was of little value for the determination of progesterone until it was shown that pretreatment of the hormone with methanolic potassium hydroxide would increase the peak one hundred-fold (143). Using a micro-cell adaptation of this method, Short and Levitt (123) were able to detect 0.05 ~g of progesterone standard. However, when samples assayed by this method and the spectrophotometric method of Short (118) were compared, the fluorescence results tended to be higher at low concentrations of progesterone and lower at high concentrations. This prompted these workers (123) to question the specificity of the fluorescence reaction, especially because of high and variable fluorescence of paper blanks. Although the theory of gas chromatography was recognized as early as 1941, it was not until 1952 that this procedure was first successfully used (72). By 1960, the technique was only beginning to be applied to steroid separation and qualitative identification (107). As recently as 1962, the first quantitative assays of progesterone by gas chromatography appeared. These were difficult, time-consuming assays with less sensitivity than available methods (51). I n 1964, the first highly sensitive gas chromatographic assay for progesterone was reported (152). The method is reported sensitive to less than 0.02 tLg of progesterone with excellent precision. Gas chromatography also appears to offer specificity that cannot be equalled by spectrophotometric or fluorometric methods. The procedure can also be adapted to isotope dilution methods for recovery estimates or quantitative measurements (22, 75, 77). The introduction of isotopic reagents for the estimation of steroid hormones has overcome the problem of inadequate sensitiviy of conventional chemical procedures. Keston et al. (76) first applied the isotope derivative principle to the estimation of amino acids in 1946,

TABLE 1 Expected sensitivity of available methods for the quantitation of progesterone Expected sensitivity (~g) Quantit~tion method

Standard cells

Spectrophotometry ~ In alcohol In sulfuric acid: ethanol Derivatives Fluorometry Gas chromatography Double-isotope derivative For a more complete listing and

2.5 2.5 2.5 0.2

Microcells 0.5 0.5 0.1-0.5 0.05

Reference

13, 34, 82, 118, 154 34, 105, 154 34, 130, 154 2, 123, 143 0.005-0.02 17, 75, 77, 152 0.005-0.02 111, 150, 151 comments on precision and recovery, see Reference 154.

318

w.R.

G O M E S AND R. E. E R B

but it was not until recently that the principle was used for steroid hormones (11, 14, 70). The general method entails addition of an isotopically labeled steroid (e.g., C~-labeled) to the unknown sample. The endogenous steroid is extracted and pm'ified in a mixture with the labeled compound, and both are converted to a derivative, using a reagent labeled with a second isotope (e.g., Hs). The amount of H ~ in the final isolated derivative quantitates the amount of derivative formed and the C" measures procedural losses. This procedure appears to be limited in sensitivity only by the specific activities of the derivative-fornfing reagent and the labeled steroid. The double-isotope derivative principle was first applied to the estimation of aldosterone (11) and testosterone (70) by forming the acetate of these hormones using acetic anhydrideI t ~ and C~'-labeled steroids. This method could not be applied per se to the estimation of progesterone, since the hormone cannot be aeetyluted; therefore, it was necessary to investigate other derivatives. Riondel et al. (111) first applied the double-isotope derivative principle to progesterone estimation using progesteroneH ~ with thiosemicarbazide-S~ as the derivativeforming reagent. A later report by Woolever and Goldfein (151) advocated the conversion of progesterone to 20 fl-ol, using tritiated sodium borohydride and progesterone-C ~ as the isotope sources. This method is reported sensitive to 0.01 tLg of progesterone or less (150), and appears highly specific and adequately precise (151). E V A L U A T I O N OF ]~IETtIODS AND P O S S I B I L I T I E S

made by the conversion of progesterone to a more highly fluorescent compound such as Finkelstein et al. (44) reported for estimation of testosterone. Judging from the very r a p i d advances in gas chromatography, one must expect the excellent method of Yannone et al. (152) to be superseded soon by even more sensitive (22), equally specific methods. The developmental possibilities for gas chromatography in qualitative and quantitative steroid assays are extensive. Although the double-isotope derivative methods currently available for progesterone research are extremely sensitive (Table 1), it is not unreasonable to believe that sensitivity can be further increased tenfold or more. Use of borohydride of a higher specific activity (50 mC/mM was used in the original study--200 mC/mM and higher is now available) could, in itself, increase sensitivity remarkably. Other developments might include the chemical (103) or enzymatic (67) conversion of progesterone to a hydroxylated derivative (e.g., 20 fl-ol), followed by aeetylation with labeled acetic anhydride. The analysis could then be carried out with higher recovery, using a conversion reagent avaiIa'ble with a very high specific activity. A less likely possibility is the conversion of progesterone to 20 fl-ol by 20 fl-hydroxysteroid dehydrogenase (67), using tritium-labeled reduced nicotinamide adenine dinucleotide ( N A D H ~) as a tritium donor. This conversion should be quantitative, but one would likely have to prepare N A D H ~ in the laboratory (1) and the compound would probably fail to meet the specific sctivity requirements.

FOR F U T U R E DEVELOPMENT

A major problem in progesterone research has been to develop methods with greater sensitivity without losing reliability. As shown in Table 1, methods employing spectrophotometric end points are sensitive only to 2.5 t~g of the hormone unless micro-ceils are used. This adaptation increases sensitivity fivefold, but also magnifies the problem of interference from impurities. Barring new developments in ultraviolet instrumentation, or discovery of a new color-forming reagent, it appears that the spectrophotometric methods have nearly achieved their peak sensitivity. These methods, however, should continue to be of great value where extreme sensitivity is not the p r i m a r y criterion. The fluorometric procedure used by Short and Levitt (123) is quite sensitive (Table 1) but requires adequate purification and extreme care with reagents and p a p e r to insure the necessary specificity. F u r t h e r advances might be

PROGESTINS

IN

THE

FtOVINE

SOURCES AND ENDOGENOUS COMPOUNDS

E d g a r (35), in 1953, appears to have been the first to identify progesterone in the bovine corpus luteum (CL). Following gonadotrophin treatment of a calf, he isolated 20 tLg of progesterone from 60 g of luteal tissue. A year later, t I a y a n o et al. (66) demonstrated the conversion of progesterone to 20 fl-ol by bovine CL in vitro; and, in ]957, Zander et al. (155) isolated 20 a-ol and 20/~-ol from human ovaries. In 1958, Gorski et al. (57) reported the isolation of progesterone and 20 fl-ol from ovaries of nonpregnant cows. A third ultraviolet absorbing area, corresponding to 20 a-ol or A~androstene-3, 17-dione, was noted, but insufficient amounts were present for identification. In a subsequent publication, Gorski et al. (58) demonstrated the presence of progesterone and 20 fi-ol in corpora lutea, ovaries, and adrenals

PROGESTERONE

IN BOVINE REPRODUCTION

of a pregnant cow, but were unable to detect either progestin in 1,400 g of placental tissue, one liter of uterine venous blood, 305 ml of uterine venous plasma, or blood or tissues of the 258-day male fetus. Others have supported the report of progesterone and 20 fl-ol in ovarian and adrenal tissue in the bovine (9, 40, 41). Short (117) was unable to detect progesterone in 1 kg of bovine placental tissue, but Melampy et al. (93) reported high levels of a compound thought to be progesterone in bovine placenta. Later, Bowerman and Melampy (13), using a different chemical method, could detect progesterone in only one sample of eight analyzed. Progesterone and 20 fl-ol have been isolated from mare placentae (116) and progesterone and 20 a-ol from ewe placeutae (125). The 20 a-ol compound is very high in the rabbit (128), rat (17, 145), and ewe (99, 125). SYNTHESIS

AND

METABOLI~:~I OF

PROGESTERONE

I N T E E BOVINE

Rapid developments in steroid biochemistry, especially the use of radioactive tracers, have helped to elucidate the biosynthetic and metabolic pathways of progestins. Use of acetate-C 1' in in vitro incubations with bovine CL has shown that simple carbon compounds may be precursors of the progestin nucleus (136). The intermediate compounds probably include aeetoacetate, hydroxymethylglutarate, and squalene, which is a precursor of the cholestane series of compounds (96). Cholesterol is probably hydroxylated at the C-20 position and cleaved at the 2]-carbon by a bovine side-chain desmolase (137). The resulting pregnenolone is converted by two enzymes to progesterone. Duncan et al. (33) demonstrated in vitro synthesis of progesterone in swine CL and commented on sevel~l factors affecting this synthesis. Sweat et al. (136) have shown that a wide spectrum of hormones is produced from progesterone by ovarian tissue in vitro. Using C~labeled compounds, these workers reported the conversion of progesterone to both 20-hydroxy derivatives, 17-hydroxyprogesterone, 6 fl-hydroxyprogesterone, and pregnanedione. I t seems likely that more sensitive methods will demonstrate this entire spectrum and probably still more progestins in samples from endogenous sources. A f t e r progesterone enters the circulation, it is subject to metabolism in the blood. Coyle and Romanoff (24) have demonstrated the in vitro conversion of progesterone-C ~ to 20 fl-ol and three more polar compounds by bovine blood. Miller et al. (97) estimated the half-life

319

of free progesterone to be between 18 and 59 rain, with an average for 11 cows of 33.8 rain. These values must be considered maximal because progesterone-C 1~ was not isolated, but rather total radioactivity per sample was measured and reported as progesterone. Short and Rowell (126) estimated the half-life of progesterone in the blood of the ewe to be 7-8 min, using values of isolated progesterone-4-C 14. Similar studies in the human suggest a half-life of 3-5 nfin (108). Studies with progesterone-C 1' in women indicate that hydroxylated, saturated metabolites of progesterone are excreted mostly in the urine, whereas the corresponding ketonic compounds are excreted via the bile (20). I n both cases, most of the hormone is conjugated as the glucuronide. Mayer et al. (91) reported pregnanediol and two nonconjugated progesterone metabolites in sow and gilt urine, and rabbit urine has been shown to contain progesterone metabolites (19). I n contrast to these animals, progesterone metabolites have only occasionally been reported in the urine of ruminants (10). Will~ams (148) administered progesterone-4-C 1' to dairy cows and recovered only 3% of the radioactivity in urine, whereas nearly 50% was found in the feces. Boscott (12), after injecting 100 mg progesterone daily into goats, found no progestin metabolites in the urine. Other workers failed to find pregnanediol in the urine of cows. The studies of Miller and Turner (95, 96) indicate that progesterone is converted to androgenic substances in the liver and excreted via the bile into the feces. I n addition, progesterone metabolites may be excreted in the milk (148) or as carbon dioxide (54). QUANTITATIVE ESTL~[ATES OF P R O G E S T I N S

co,vs. One hesitates to summarize reported quantitative levels of progestins, because of the uncertainty of how efficiently the hormones were extracted and purified, especially in earlier studies. However, enough evidence exists to warrant doing this to show generalized trends. As shown in Table 2, the progestin levels in corpora lutea of cycling cows agree remarkably well for three of the nmre complete studies (42, 55, 89). Data from other studies support these three (47, 132, 133), but there are some conflicting data (23). In the majority of studies using bovine corpora ]urea, progesterone concentration (tLg/g) showed an initial rise during the early proliferative stages of the CL (Days 1-4 of the cycle), followed by a more or less constant level (25-35 t~g/g) until Day 9 or 10. Nonpregnant

TABLE 2 P r o g e s t i n c o n t e n t of corpus l u t e u m a a n d o v a r i a n ve i n blood p l a s m a b of t he c y c l i n g cow ( 5 5 ) a n d ewe ( 3 7 ) Ovarian vein plasma

C orpus l u t e u m

Days since estrus

Mares et al. (89) No. CL

NO. CL W t

(g)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Gomes et al. (55)

Oomes ct al. (55)

Prog.

20 fl-ol

(~g/g)

Prog.

20

fl-ol

NO. s~mples

Prog.

CL

CL W t 1.54 ¢ 1.22 ° 0.74 0.79 1.98 2.24 3.63 3.62 6.20 6.38

6.8 6.5 a 19.1 34.7 38.1 32.8 35.3 22.5 24.8

d d d 0.1 d d d 0.4 d 0.6

2 3 3 3 3 3 3 3 3 3

d d 0.9 0.2 0.9 0.9 1.4 1.3 2.1 2.5

(g)

(Pg/g)

(~g/ml)

6

0.54

24.7

1.6

5

1.88

24.7

1.6

4

4.06

25.8

1.1

4

3.70

35.0

0.6

2 3 3 3 3 3 3 3 3 3

4

5.54

33.8

1.4

1

4.29

66.4

4.2

1

4.3

4

4.20

41.9

4.1

4

5.27

44.7

6.0

4 3 2

6.70 5.83 4.32

73.2 54.9 32.2

7.0 6.1 3.0

4 3 2

3.2 6.2 2.0

4

3.53

7.0

2.1

6

3.05

14.2

4.9 2 1

1.86 2.92

10.0 d

d d

2 1

0.6 0.7

F o r a d d i t i o n a l d a t a a n d other species, see R e f e r e n c e s 13, 23, 33, 40, 41, 57, 58, 78, F i g u r e 1. b F o r a d d i t i o n a l d a t a a n d other species, see T a b l e 3. " Co rp ora l u t e a of p r e v i o u s cycle. d NO p r o g e s t i n detected.

Edgar & R o n a l d s o n (37) NO. samples

5 6 4 5 4 8 8 12 17 15 9 11 6 6 14 5 14

t'rog.

(~g/ml)

d 0.5 0.4 0.6 0.9 1.9 1.6 1.5 1.6 1.4 2.0 1.5 2.3 1.8 2.0 d

P R O G E S T E R O N E IN B O V I N E R E P R O D U C T I O N

Thereafter, the concentration of the hormone again increased to a peak on Days 14 and 15, followed by a decline to the next estrus (Table 2). Detectable progesterone is present in the regressing CL for two or three days after the next estrus period. The levels of 20 fl-ol in the CL contributed 5-20% of the total progestins, apparently depending on the method of CL collection. Since progesterone can be converted to 20 fl-ol by the incubating CL, it seems likely that collection of tissues at slaughter (42) might be expected to show higher levels of 20 fl-ol than collection from the living animal (55, 89). The data indicate that such is the case. Progestin concentrations in the CL of the bovine (Table 2) a p p e a r to be paralleled by concentrations in the ovarian venous effluent (55). Table 2 shows also the concentration of progesterone in the ovarian vein blood of the cycling cow (55) and ewe (37). Armstrong et al. (7) have shown that bovine CL exhibits maximum in vitro progesterone synthesis (270-300 tLg/g/2 hr) when the gland is removed surgically 4-13 days post-estrus. A gradual decline in synthetic capacity was noted between Days 14 and 18 post-estrus (80 tLg/g/ 2 hr). No detectable synthesis occurred with 19-day-old CL. Luteinizing hormone ( L H ) stimulated increased progesterone synthesis by all CL collected prior to 19 days post-estrus, but had no discernible effect on luteal tissue obtained on Day 19 or later. Progesterone synthesis in these latter CL's could be only part i a l l y restored by addition of pregnenolone or glucose-6-phosphate plus NADP* to the incubation medium (7). Duncan et al. (33) reported the in vitro synthesis of progesterone by slices of porcine CL. As with the bovine CL (7), glands collected 4-16 days post-estrus were able to synthesize progesterone, but those obtained on Day 18 contained no detectable progesterone before or after incubation. I t can be generalized from several studies that growth in weight of the CL and progesterone content, synthesis, and secretion are closely related to the degree of luteinization as determined by histological studies (26, 89). During the first five days of the cycle, the granulosa cells of the CL are gradually inteinized (26), the CL grows in weight (55, 89), and progesterone content and synthesis become detectable (7, 55, 89). A f t e r Day 6, the theca interna cells become luteinized (26), the CL grows larger, and progesterone concentration is higher (55, 89). Degeneration of the luteal cells begins on Day 16 or 17 in the nonpregnant bovine (26, 89), corresponding to a decrease in weight and progesterone concentration (55,

321

89), rate of release (55), and a lowered capacity to synthesize (7). Similar changes in R N A / D N A ratios and per cent Type I and Type I I cells (46) were shown by Mares et al. (89). I n a study designed to compare luteal and blood plasma progestins in the nonpregnant bovine, Gomes et al. (55) showed a significant correlation between CL progestins (t~g/g or tLg/CL) and ovarian vein progestin concentration (#g/ml of plasma). However, they reported that the peripheral blood progesterone levels found in their study did not reflect the stage of the estrous cycle, quantity of concentration of progestin in the CL, or concentration of progestin in ovarian vein blood plasma. Ovarian vein progestin concentrations in the cow and ewe show similar trends (Table 2) and indicate that CL levels of the h o l ~ o n e may adequately reflect secretion rates. The work of Stormshak et al. (135) in the cycling ewe supports the hypothesis that luteal progestin content is indicative of the amount of hormone released into the blood, but Clegg et al. (23), in a study involving only seven cows, suggest that CL levels of progesterone do not reflect secretory rate. I t is discouraging to note that Gomes et al. (55) found no relationship between tissue or ovarian vein plasma progesterone and peripheral blood plasma levels of the hormone. I t should be pointed out, however, that micro-cell attachments were not used for the spectrophotometric quantitative step (55). Thus, readings were often made near the limit of sensitivity (Table 1), increasing the possibility of error. ~¢[cCraeken (86) has shown that peripheral blood plasma progesterone levels begin to decrease within 15 rain after CL removal and fall to nondetectable levels within 24 hr, indicating that a positive relationship may exist between ovarian and peripheral levels of the hormone. This would suggest that a highly sensitive assay allowing analysis of multiple samples from one animal throughout the cycle might yield results which would lead to a better understanding of ovarian-peripheral progesterone relationships. P r e g n a n t cows. Figure 1 was drawn in an attempt to generalize available data on progestin levels in corpora lutea and peripheral blood plasma of pregnant cows. CL data are combined from the studies of Zimbelman et al. (158-161, control data), Stormshak and Erb (134), Estergreen et al. (43), and Gomes et ah (56). The data on peripheral plasma hormone levels are taken from the reports of Short

322

w.R.

G O M E S A N D R. E, E R B

00 .J O. 2 D .J ,<

•

(/)

a. 4 0 nO

A

E LIJ

-1i1

CORPORA LUTEA °

[] PERIPHERAL PLASMA b

_

o b.

o

(.9

\

E 1.5 "' D. .J

30

O

o

(.9

1.0

:k 20

!

Or)

ul

I-Or)

0.5 ~

Z

'"

o 0t~

o

I0

I---

{1.

o

0

I 8

I 16

I I 2J4~'(~0

I

I

I

I

I

0

o_

220 260 I00 140 180 DAYS SINCE ESTRUS Fro. 1. Progestin (progesterone and 20 fl-ol) in corpora ]utea and peripheral blood plasma of pregnant cows. References 40, 41, 43, 56, 134, 158-161. h References 13, 56, 87, 117. 0

(119), Bowerman and Melampy (13), Gomes et al. (56), and MeCracken (87). CL levels of progesterone, both in total (t~g/CL) and in concentration (tLg/g), are in excellent agreement for several studies (56, 134, 158-161). Others (]3, 93) tend to report lower quantitative values; therefore , these are not shown on the curve in Figure 1. Of the combined data in the CL curve in Figure 1, the values of control cows reported by Zimbelman et al. (158-161) tend to be lower; but the differences are not large when the other data are restricted to cows under 10 yr of age. Since earlier studies have shown very close parallelism between total progesterone in the CL and progesterone concentration (40, 41), only the latter has been shown in Figure 1. I n several studies, essentially no difference was found in CL progestins 14-16 days following estrus in pregnant or nonpregnant cows (40, 47, 134). Pregnant cows show a slight decline by Day 19, but the most pronounced decline occurs after Day 42. A leveling off is seen after Day 56 and a further decline at about 120 days of pregnancy. A later rise reaches a peak at about 250 days, followed by a decline to the time of parturition (Figure 1). Similar changes are seen in peripheral blood progesterone levels (Figure 1). Gomes et al. (56) have shown that ovarian vein plasma

progestin concentration also declines after 250 days in the bovine. This concentration reaches undetcctable levels after parturition (43). Progestin was also undetectable in a CL obtained eight days post-partum (134). Edgar and Ronaldson (37) reported that ovarian vein blood progesterone remained at about 1.5 t'g/ ml during Days 7-28 of pregnancy in the ewe, then increased to over 2 tLg/ml on Day 35. Thereafter, the concentration remained near 1.5 t*g/ml until Day 111, followed by a gradual decline to nondetectable levels at 140 days. These workers (37) suggested, from their data, that uterine contents in the pregnant ewe are not an important source of progesterone. However, other work has shown that the ewe does not abort following ovariectomy after Day 60 of pregnancy (18), peripheral blood progesterone levels in the pregnant ewe do not fall after ovariectomy, and the placenta of the ewe has been found to be a source of progesterone and 20 a-ol (125). Other species. Although this paper is primarily concerned with bovine progestins, and it is often difficult to extrapolate between species, a certain amount of reference to comparative data may be helpful in elucidation of the role(s) of progesterone in reproductive processes. For this reason, a summary of progesterone levels in the blood of a number of species

PROGESTERONE

is shown in Table 3. These d a t a have viously been s u m m a r i z e d in p a r t (35, 41, F o r tissue a n d follicular fluid levels of hormone, the r e a d e r is r e f e r r e d elsewhere 40, 41, 113).

IN BOVINE

pre54). the (35,

PROGESTIN AND NOR]'C[AL CYCLIC FUNCTION O b s e r v a t i o n s f r o m several e x p e r i m e n t s indicate t h a t p r o g e s t e r o n e is associated w i t h the a v a i l a b i l i t y o f L H d u r i n g late p r o e s t r u s or

early d i e s t r u s (40). M e C a n n (84) f o u n d t h a t p r o g e s t e r o n e o r estradiol b e n z o a t e alone h a d little effect on L I t release in the ovariectomized rat, b u t p r o g e s t e r o n e i n j e c t i o n s into estrogenp r i m e d females reduced p l a s m a L H to nondetectable levels. As reviewed earlier b y E r b (40), r a t i o s of p r o g e s t e r o n e to estradiol of 12.5:1 to 7 5 : 1 a p p e a r to be o p t i m a l f o r the i n d u c t i o n o f estrus in the ovarieetomized cow, but ratios h i g h e r t h a n 7 5 : 1 will block this

TABLE 3 A summary: L e v e l s o f p r o g e s t e r o n e i n b l o o d o r p l a s m a Species Cow a

Ewe a

Reproductive status Preovulatory-1 day Cycle-12 days -12 days - 1 day -13 days - 1 day -2-5 days -6-8 days -9-15 days -19-20 days P r e g n a n t - 9 0 days -10-49 days -50-89 days -90-]29 days -130-169 days -170-209 days -210-249 days -250-280 days -111-117 days -250-254 days -281-282 days Cycle Mid-htea] Late luteal P r e g n a n t - 6 days -1-4 wk -5-6 wk -7-16 wk -17-18 wk -19 wk -20 wk -15 days -60 days -96 days -99 days

323

REPRODUCTION

as d e t e r n f i n e d b y c h e m i c a l a s s a y

Fraction of blood Peripheral plasma

Peripheral blood

Ovarian plasma

Ovarian plasma Ovarian blood

Peripheral plasma

Progesterone

(~g/lO0 ml) Reference 0.20 0.19 0.92 Not detected 1.06 0.71 0.36 0.46 1.68 0.09 1.18 0.9 1.4 0.4 3.1 3.4 4.0 3.1 264 259 140

95 104 125 130 215 160 125 40 Not detected 0.43 Not detected 0.68 0.91

Peripheral plasma

0.75 1.70 3.]2 2.92 2.04 0.91 1.04 1.20 0.34 0.67 0.49

P r e g n a n t - 1 3 4 days Ovarian blood Peripheral plasma -112 days * For additional data, see Table 2 and Figure 1.

230 0.71

Sow

Goat

Cycle-Mid-hteal P r e g n a n t ~ 0 days -48 days -49 days -53 days -55 days -99 days -102 days -112 days -113 days -114 days

117 117 86 87 87 55 55 55 55 55 87 93 93 93 93 93 93 93 54 56 56 124 124 85 37 37 35,37 37 37 37 99 117 117 !17 117,120 120 120 120 120 120 120 120 120 120 ]20 109 117

324

w.R.

G O M E S AND R. E. E R B

condition (92). I n intact cows, 10 mg of progesterone administered during estrus reduces the time to next ovulation by about 10 hr (62). W o r k with the bovine and the rat indicates that progesterone accomplishes this end by enhancing the hypothalamo-hypophyseal release of ovulating hormone (likely L H ) . Foote et al. (47) observed a high incidence of cystic ovaries following supra-vaginal removal of the CL on D a y 14 of the cycle. Staples et al. (132) produced cystic ovaries in significant proportion of heifers when CL inhibition and precocious ovulation were induced with oxytocin. Conversely, 50 mg of progesterone daily will inhibit ovulation and estrus effectively, but large follicles still appear on the ovary (144). This suggests that a minimal level of progesterone is necessary to enhance release of gonadotrophin, but a higher level may be inhibitory. The inhibition and synchronization of estrus and ovulation in cattle by progesterone and other progestins have been widely used for both practical and experimental reasons. The practica~ aspects have been adequately reviewed elsewhere (49, 61), but the value of such compounds in the experimental nmnipulation of cyclic functions is worthy of note here. The injection of repositol progesterone (76 mg/cwt) into heifers on the day of heat or on D a y 8 or 16 of the cycle significantly decreased pituitary levels of follicle-stimulating hormone ( F S H ) and L H and reduced the diameter of the CL. When progesterone (100 mg/ewt) was injected on D a y 1 or Day 4 of the cycle, CL weight, progesterone content, and functional cells in the CL were reduced when inspected on Day 14; but estradiol had no a p p a r ent effect on the total amount of progesterone in the CL (83). This a p p a r e n t luteolytic--or anti-luteotrophic--effect of progesterone offers a basis for the study of CL growth and maintenance. I f one were to determine which gonadotrophin(s) were decreased in bovine pituitary effluent (30) following progesterone administration, it might be possible to determine t h e nature and normal level of the pituitary luteotrophin in the bovine and to add to our meager knowledge of progesterone-(uterine) -hypothalamo-hypophyseal-owrian relationships. Such studies might also be helpful in determining whether other progestins act in the same manner as progesterone itself (106). CORPUS L U T E U M

MAINTENANCE

The most important factor in the onset of pregnancy, other than the presence of a zygote, is maintenance of the CL. The mechanism of

this maintenance or, conversely, the mechanism of luteolysis in the nonpregnant bovine, is poorly understood. Our knowledge of these processes is most advanced for the rat and mouse; but it appears that prolactin, the luteotrophic hormone ( L T H ) in these species, is ineffective for CL maintenance in other species (122). As mentioned earlier, Hansel and Wagner (62) reported the anti-]uteotropic nature of oxytocin in the bovine. Simmons and Hansel (129), in an ingenious experiment using the prevention of this effect as an end point, reported that a luteotrophic factor is present in crude anterior pituitary extracts of the bovine. The oxytocin-induced effect was also overcome by human chorionic gonadotrophin (HCG), but was unaffected by bovine growth hormone, ovine prolactin, or equine LH. I t is disappointing that bovine L H was not available for this study (129), since H C G exhibits predominantly L H action (157), and ovine L H increased the in vitro synthesis of progesterone synthesis in human CL, as does HCG, but ovine L H is ineffective (110). A n y theory of CL maintenance or luteolysis must necessarily include a uterine effect, since hysterectomy will result in CL maintenance (5), oxytocin repression of the CL fails in the hysterectomized cow (133), and insertion of foreign bodies into the uterus can cause precocious estrus and ovulation (62). These results suggest that a certain normal uterine condition is necessary during the early cycle for continued secretion of L T H in the bovine. Oxytocin, hysterectomy, or foreign objects disr u p t this necessary condition. I f a zygote does not enter the uterus, a substance from that organ may induce luteolysis, either by interrupting pituitary L T H release or by a direct LTH-over-riding effect on the ovary (122). PROGESTI~qS A:ND PREGNANCY Essentiality of the CL. As reviewed earlier (41), the CL appears essential throughout pregnancy in the rabbit, rat, sow, goat, and bitch, but for less than full term in primates, mares, ewes, and guinea pigs. In all of the species studied to date, however, progesterone or its equivalent activity is required for the normal span of pregnancy. In the cow, the necessity of the CL during pregnancy remains controversial. I t is generally concluded that the CL is required for about 200 days in this animal, but in one study removal of the CL after 200 days did not result in abortion prior to 250 days (88). I n another study, when cows were ovariectomized after 200 days, eight of nine aborted between 238 and 274 days of

PROGESTERONE IN BOVINE REPRODUCTION pregnancy (43). Furthermore, peripheral blood progesterone concentrations dropped to undetectable levels a f t e r ovariectomy (56), whereas this operation does not lower the hormone levels in peripheral plasma of the p r e g n a n t ewe. As pointed out earlier in this paper, attempts to isolate progesterone f r o m bovine placenta have generally been unsuccessful (58, 117), but occasional positive results have been noted (13, 43, 93). F r o m the above evidence, one might propose the following hypothesis to explain the sources of progesterone in p r e g n a n c y nmintenance in the bovine : The m a j o r source of progesterone throughout pregnancy in the cow is the CL (58), but ovaries and adrenals might contribute a significant amount (58, 134) and body fat may store the hormone to be used if needed (87). The placenta in the cow produces a v e r y low level of progesterone. I n most cases, this level is insufficient to maintain p r e g n a n c y following ovariectomy but m a y be adequate when only the CL is removed. Because of individual variation in placental production of the hornione, an occasional cow may fall to abort following ovariectomy. This postulated low level of progesterone synthesis is not unique among animals. I f fetuses are removed f r o m the rat in such a way that the placentae remain intact, 12 placentae will maintain one fetus in the ovarieetomized rat (65). The general failure to find progesterone in bovine placenta cannot be considered p r o o f of an absence of the hormone, since the levels may be so low as to have escaped detection by the methods used but high enough to exert an i m p o r t a n t local uterine effect (25, 50). Use of more sensitive assays (Table 1) should enable one to better resolve this question. REFEREI~CES

(1) ABRAHAM, S. 1964. Biosynthesis of Labeled Carbohydrates and Other Compounds of Biochemical Interest. Atomlight, 36: 1. (2) AB~A]~AM, R., AND STAUDINGER, II. 1964. Spektorscopische Untersuchungen zur Fluorometrischen Steroidanalyse. Z. Klin. Chem., 2: 16. (3) AMERICAN MEDICAL ASSOCIATION COUNCIL ON PHARI~ACY AND CHEMISTRY. 1936. J. Am. Med. Assoc., 106: 1808. (4) ANDERSON, F. O., CRISP, L. R., RIGGLE, G. C., VERUK, G. G., HEFTMANN, E., JOHNSON~ D. F., FRANCOIS~ D., AND PERRINE, T.D. 1961. Steroid Analyzer. Anal. Chem., 33 : 1606.

325

(5) ANDERSON, L. L., BOWERMAN, A. M., AND MELAMPY, R. M. 1963. Neuro-Utero-Ovarian Relationships. I n A. V. Nalbandov, Ed. Advances in Neuroendocrinology. University of Illinois Press, Urbana. (6) ANDERSON, L. L., NEAL, F. C., AND MELAMPY, R. M. 1962. Hysterectomy and Ovarian Function in Beef Heifers. Am. J. Vet. Research, 23: 794. (7) ARMSTRONG, D. T., BLACK, D. L., AND CONE, C. E. 1964. Metabolic Studies with Active and Involuting Bovine Corpora Lutea. (Abstract.) Federation Proe., 23: 462. (8) AXELROD, L. R., AND WERTHESSEN, N. T. 1961. Blood as a Site for the Conversion of the Steroid Hormones. Endocrinology, 68:180. (9) BALFOUR, W. E., COMLINE, R. S., AND SHORT, R. V. 1959. Changes in the Secretion of 20 a-Hydroxypregn-4-en-3-one by the Adrenal Gland of Young Calves. Nature, 183: 467. (10) BENSON, G. K., AND COWIE, A. T. 1957. Reviews of the Progress of Dairy Science. Section A. Physiology of Dairy Cattle. Hormones in Reproduction and Lactation. J. Dairy l%search, 24: 252. (11) BOJESON, E., AND DEGN, H. 1963. A Double Isotope Derivative Method for the Determination of A]dosterone in Peripheral Plasma. Acta Endocrinol., 37: 541. (12) BOSCOTT, R. J. 1952. The Metabolism of Progesterone in the Goat. The Separation of Urinary Cortical Steroids. Colloq. on Endocrinol. CIBA Found., 2: 327. (13) BOWERMAN, A. M., AND MELAMPY, R. M. 1962. Progesterone and A~-Pregnen-20 fl-ol3-one in Bovine Reproductive Organs and Body Fluids. Proc. Soc. Exptl. Biol. Med., 109 : 45. (14) BURGER, H. G., KENT, J. R., AND KELLIE, A . E . 1964. Determination of Testosterone in Human Peripheral and Adrenal Venous Plasma. J. Clin. Endoerinol. Metabolism, 24:432. (15) BURTON, R. B., ZAFPARONI, A., AND KEUTMANN~ E. ]:I. 1951. Paper Chromatography of Steroids. II. Cortieosteroids and Related Compounds. J. Biol. Chem., 188: 763. (16) BusH, I. E. 1961. Chromatography of Steroids. Pergamon Press, New York. (17) CARLSON, I. It., BLAIR, A. J., AND MEYER, R. K. 1964. Analysis of Rat Ovarian Venous Effluents by Gas-Liquid Chromatography. (Abstract.) Proc. AmL Meet. Endocrine Soc. (18) CASIDA, L. E., AND WARWICK, E. J. 1945. The Necessity of the Corpus Luteum for Maintenance of Pregnancy in the Ewe. J. Animal Sci., 4: 34. (19) CHAMBERLAIN, J., KNIGHTS, B. A., AND THOMAS, G. H. 1963. Analysis of Steroid

326

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

(28)

(29)

(30)

(31)

(32)

(33)

(34)

w. 1~. GOMES AND I%. E. ERB Metabolites by Gas Chromatography. J. :Endocrinol., 26 : 367. CtIANG, :E., SLAUNWttITE, W. R., AND SAND° FEral, A. A. 1960. Biliary and Urinary Metabolites of 4-C~-Progesterone in Human Subjects. J. Clin. :Endocrinol. Metabolism, 20 : 1568. CI~ANNING, C. P., AND VILLEE, C. A. 1964. Some :Effects of Luteinizing ttormone on Metabolism of the Corpus Luteum of the Rat. (Abstract.) Proc. Ann. Meet. Endocrine Soc. CLARK, S. J., AND WOTIZ, H. tI. 1963. Separation and Detection of Nanogram Amounts of Steroids. Steroids, 2:535. CLEGG, M. T., SQUIRES, C., AND GANONG, W . F . ]962. Progesterone Output from Cow Ovaries. (Abstract.) Proc. Ann. Meet. :Endocrine Soc. COYLE, M. G., AND R0~ANOPF, :E. B. 1964. Transformation of 4-C~-Progesterone by Bovine Blood. (Abstract.) Federation Prec., 23: 462. CSAFO, A. 1961. The Role of Progesterone in the Maintenance and Termination of Pregnancy. I n A. C, Barnes, Ed. Progesterone. Brook Lodge Press, Augusta, Michigan. CUFFS, P. T., LABEN, R. C., AND MEAD, S. W. 1959. Histology of Pituitary, Adrenal, and Reproductive Organs in Norreal Cattle and Cattle wi~h Lowered Reproductive Efficiency. Hilgardia, 29: 383. DANIEL, 5. C., JR., AND LEVY, J. D. 1964. Action of Progesterone as a Cleavage Inhibitor of Rabbit Ova in Vitro. J. Reprod. Fertil., 7: 323. DEVENUTO,F., MULE, S., AND WESTPItAL, U. 1958. Perextraction, A Procedure to Extract C_~ Steroids from Tissues and Blood. Arch. Biochem. Biophys., 73:451. DOI~INGUEZ, O. V., SEELY, ,]-. R., AND GORSKI, J. 1963. Studies on the Ace~ylation of Steroids Using l-Carbon-14-Acetic Anhydride. Anal. Chem., 35: 1243. DONALDSON, L. E., AND HANSEL, W. 1964. Collection of Blood from the Cavernous Sinus in the Cow. J. Dairy Sei., 47: 98. DONKER, J. D., NICHOLS, J. R., GRAHAIV[, :E. F., AND PE~ERSON, W. E. 1958. Controlled Estrus in Cattle. ProF. I I I Sympos. Reprod. and InfertiI. Pergamon Press, New York. DORF!~[AN,R. I., I~INCL, F. A., AND RINGOLD, H. J. 1961. Anti-Estrogen Assay of Neutral Steroids Administered by Gavage. Endocrinology, 68: 43. DUNCAN, G. W., BOWERMAN, A. N., ANDERSON, L. L., HEARN~ W. R., AND MELA1VIFY~ R. M. 1961. Factors Influencing in Vitro Synthesis of Progesterone. :Endocrinology, 68 : 199. DUSZA, J. P., HELLEI~, M., AND BERNSTEIN, S. 1963. U]travlolet Absorption. I n L. L.

(35) (36)

(37)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

(45)

(46)

(47)

(48)

(49)

Engel, : E d . Physical Properties of the Steroid Hormones. MacMillan Co., New York. :EDGAR,D. G. 1953. The Progestin Content of Body Fluids. J. Endocrinol., 10:54. EDGAR, D. G., FLUX, D. S., AND RONALDSON, J. W. 1959. Comparison of Chemical and Biological :Estimations of Plasma Progestin. J. Endocrinol., 19: 44. :EDGAR, D. G., AND RONALDSON, J. W. 1958. Blood Levels of Progesterone in the :Ewe. J. :Endoerinol., 16: 378. EMMENS, C. W. ]959. Role of Gonadal Itormones in Reproductive Processes. In H. H. Cole and P. T. Cupps, Eds. Reproduction in Domestic Animals. Vol. I. Academic Press, New York. ENGEL, L. L., A~]) CARTER, P. 1963. Partition Coefficients. I n L. L. :Engel, Ed. Physical Properties of the Steroid Hormones. MacMillan Co., New York. :EBB, R. E. 1961. Significance of Female Sex Steroids in Bovine Reproduction. V. Biennial Sympos. Animal Reprod., Knoxvi]le, Tenn. EBB, R. :E., AND GOMES, W. R. 1962. Cyclic Changes in Levels of Female Sex Steroids. Sympos. No. Atlantic See., Am. Soc. Animal Sci., Princeton, N. J. EBB, R. E., AND STORMSttAK, F. 1961. Progestins in Corpora Lutea, Ovaries, and Adrenals A f t e r :Estrus and Breeding of l~ormal and Abnormal Cows. J. Dairy Sci., 44 : 888. :ESTERGREEN, V. L., JR., GORES, W. R., FROST~ 0. L., AND :EBB, R. :E. 1964. Unpublished data. FII~KELSTEIN, M., FORCIt~ELLI, :E., AND DOlCF~AN, R. I. 1961. Estimation of Testosterone in Human Plasma. J. Clin. :Endocrinol. Metabolism, 21: 98. FroST, N. L., S ~ T ~ A N , F. W., RmOR, E. M., AN]) CASIDA, L. E. ]963. Factors Affecting Ovulation and Follicular Cyst Formation in Sows and Gilts Fed 6-Methyl17-Acetoxyprogesterone. J. Animal Sci., 22 : 66. FOLEY, R. C., AND GREENSTEIN, J. S. 1958. Cytological Changes in the Bovine Corpus Luteum During Early Pregnancy. Proc. I I I Sympos. Reprod. ]nfertil. Pergamon Press, New York. FOOTE, W. D., ZIMBEL~AN, R. G., LOY, R. G., AND CASIDA, L. :E. 1959. Endocrine Activity of Corpora Lutea from First-Service and Repeat-Breeder Dairy I~eifers. J. Dairy Sei., 42: ]944. FRANCOIS, D., JOHNSON, D. F., AND HEFT~[ANN, E. 1963. Programmed Gradient :Elutlou Chromatography with the Steroid Analyzer. Anal. Chem., 35: 2019. FRIDI~IANDLER, L., AND PINCUS, G. 1964. Pharmacology of Reproduction and Fertility. Ann. Rev. Pharmacol., 4: 177.

PROGESTERONE IN BOVINE REPRODUCTION (50) FVCHS, A. R. 1964. Effect of Intra-Amnionic Administration of Progesterone on Oxytocin-Induced Labour in ]~abbi%s. Aeta Endocrinol., 46: 235.

(51) FUTTERWEIT, W., McNIvEN, N. I., AND I)ORFMAN, R. I. 1963. Gas-Chromatographic Identification of Progesterone in Human Pregnancy Plasma. Biochim. et Biophys. Aeta, 71: 474. (52) GARDNER., M. L., FIRST, N. L., AND CASIDA, L . E . 1963. Effect of Exogenous Estrogens on Corpus Luteum Maintenance in Gilts. J. Animal Sci., 22:132. (53) GLASSER, S. R., AND SOUPART, P. 1964. Actinomycin D Effects on Implantation and Decidual Cell Response. (Abstract.) Proe. Ann. Meet. Endocrine Soc. (54) GOMES, W. 1~. 1962. Assay for Progestins in Peripheral and Ovarian Venous Blood from Cows. Thesis, Washington State University. (55) GOMES, W. R., ESTER~REEN, V. L., JR., FRost, O. L., ANI) EzB, i~. E. 1963. Progestin Levels in Jugular and Ovarian Venous Blood, Corpora Lutea, and Ovaries of the hIonpregnant Bovine. J. Dairy Sci., 46: 553. (56) GOMES, W. R., FROST, O. L., AND ESTER0REEN, V. L., JR. 1962. Progestins in Ovarian and Peripheral Blood of Cows During Late Pregnancy. J. Dairy Sci., 45: 670. (57) GORSKI, J., DOMINGUEZ, O. V., SAi'~UELS, L. T., AND ERB, R. E. 1958. Progestins of the Bovine Ovary. Endocrinology, 62: 234. (58) GORSKI, J., EEB, R. E., DICKS01q, W. M., AN]) BUTLER, tI. C. ]958. Sources of Progestins in the P r e g n a n t Cow. J. Dairy Sei., 41 : 1380. (59) GORSKI, J., AND 1N"ICOLETTE, J. A. 1963. Early Estrogen Effects on Newly Synthesized RNA and Phospholipid in Subcellular Fractions of Ra¢ Uteri. Arch. Biochem. ]3iophys., 103: 418. (60) HANSEL, W. 1961. The Hypothalamus and Pituitary Function in Mammals. Intern. J. Fertil., 6: 241. (61) HANSEL, W., VAN BLAKE, H., AND MALYEN, P. 1964. Artificial Estrous Cycle Synchronization in Cattle by Feeding Techniques. Proc. Cornell Nutr. Conf. for Feed Manufacturers. (62) HANSEL, W., AND WAGNEI~ W. C. 1960. Lutea] Inhibition in the Bovine as a Result of Oxytocin Injections, Uterine Dilatation, and Intrauterine Infusions of Senfinal and Preputial Fluids. J. Dairy Sci., 43: 796. (63) I-IARA, S., TANAKA, ]:L, AND TAKEUCttI, M. 1964. Systematic Analysis of Thin-Layer Chromatogram. Chem. Pharm. Bull., 12: 626. (64) I'IARTMAN, C. G. 1960. Physiological Mechanisms Concerned with Conception--An Inventory of Unanswered Questions. Pers. Biol. Med., 4: 77.

327

(65) HATERIUS, H. O. 1936. Reduction of Litter Size and Maintenance of Pregnancy in the Oophorectomized Rat: Evidence Concerning the Endocrine Role of the Placenta. Am. J. Physiol., 114: 399.

(66) HAYANO,

(67)

(68)

(69)

(70)

(71)

(72)

(73)

(74)

(75)

(76)

(77)

(78)

(79)

M., LINDBERG, M. C., WIENER,

M.,

ROSENKRANTZ~ i . , AND DORFMAN, R. I. 1954. Steroid Transformations by Corpus Luteum Tissue. Endocrinology, 55: 326. HENNING, H. D., AND ZANDE4¢, J. 1962. Verwendung von Hubeners 20 fl-Hydroxysteroid-Dehydrogenase bei mikrochemischer Identifizierung und Trennung yon Steroiden. Z. Physiol. Chem., 330: 31. I'IILLIARD, J., ARCHIBALD, D., AND SAWYER, C. H. 1963. Gonadotropic Activation of Preovulatory Synthesis and Release of Progestin in the Rabbit. Endocrinology, 72: 59. It0LM, L. W., AND SHORT, R. V. 1962. Progesterone in the Peripheral Blood of Guernsey and Friesian Cows During Prolonged Gestation. J. Reprod. Fertil., 4: 137. HUDSOn, B., COG~LAN, J., DULM~NIS, A., WINTOUR, M., AND EKKEL, I. 1961. The Estimation of Testosterone in Biological Fluids. Australian J. Exptl. Biol. Med. Sei., 41: 235. JAILER, J. W. 1948. A Fluorometric Method for the Clinical Determination of Estrone and Estradiol. J. Clin. Endoerino]. Metabolism, 8: 564. JAMES, A. T., AND MARTIN, A. L. P. 1952. Gas-Liquid Partition Chromatography: The Separation and Micro-Estimation of Volatile F a t t y Acids from Formic to Dodecanoic Acid. Biochem. J., 50: 679. JOHNSOh~, K. R., AND ERR, R. E. 1962. Maintenance of Pregnancy in Ovariectomized Cattle with Progestin Compounds and Their Effect on Progestin Levels in the Corpus Luteum. J. Dairy Sci., 45: 633. KARLSON, P. ]963. New Concepts on the Mode of Action of Hormones. Pers. Biol. Med., 6: 203. KARMEN, A., MCCAFFREY, I., AND KLIMAN, B. 1962. Derivative Ratio Analysis: A New Method for Measurement of Ster()ids and Other Compounds Using Radioassay by Gas-Liquld Chromatography. A~ml. Biochem., 6: 31. I~ESTON, A. S., UNDEFRIEND, S., AND CANNON, R. K. 1946. Microanalysis of Mixtures (Amino Acids) in the Form of Isotopic Derivatives. J. Am. Chem. Soc., 68: ]390. KLII~AN, B., AND BRIEFER, C., JR. 1964. Gas-Liquld Chromatography of Carbon-14 and Tritium Labeled Steroids. (Abstract.) Froc. Ann. Meet. Endocrine Soc. ]~RISTOFFERSEN, J. 1960. Ges~cogens in Corpus Luteum of Cattle. Acta Endocrinol., 33 : 417. I~U~AR, D., AND BARNES, A. C. 1963. Studies on the Mechanism of Action of

328

W. 1%. GOMES AND R. E. ERB

Progesterone on the Human Myometrium. Bull. Johns Hopkins Hosp., 113: 330. (80) LAURITZEN, C. 1963. Biologische Wirkung des 20 fl-Hydroxy-Pregn-4-en-3-on. Acta Endocrinol., 44: 225. (81) LISEOA, B. P. 1963. Separation and Charaeterisation of A*-3-Ketosteroids of the Pregnane Series by Means of Thin-Layer Chromatography. Acta Endocrinol., 43:47.

(95)

(96)

(82) LOY, R. G., MOSIIAN, W. H., AND CASIDA,

(83)

(84)

(85)

(86)

(87)

(88)

(89)

(90)

(91)

(92)

(93)

(94)

L . E . ]957. A Rapid Method for Purification and Quantitative Estimation of Progesterone from Luteal Tissue. J. Biol. Chem., 229 : 583. LoY, R. G., ZIMBELMAN, i~. G., AND CASIDA, L. E. 1960. Effects of Injected Ovarian Hormones on the Corpus Lutemn of the Estrual Cycle in Cattle. J. Aninlal Sei., 19 : 175. McCANN, S. M. 1962. Effect of Progesterone on Plasma Luteinizing Hormone Ae%ivity. Am. J. Physiol., 202:601. MOCLURE, T. J., AND RONALDSON, J. W. ]962. Progesterone Levels in the Ovarian Venous Plasma of Ewes with Abnormal Ova. New Zealand J. Agr. Research, 5: 507. MCCRACKEN,J. A. 1963. Plasma Progesterone Concentration A f t e r Removal of the Corpus Luteum in the Cow. Nature, 198: 507. McCRACKEN, J. A. 1964. Progesterone in the Body F a t of the Dairy Cow. J. Endocrinol., 28 : 339. McDoNALD, L. E., MCNUTT, S. H., AND NICHOLS, R. E. 1953. On the Essentiality of the Bovine Corpus L~teum of Pregnancy. Am. J. Vet. Research, 14: 539. MARES, S. E., ZIMBELMAN, R. G., AND CASIDA, L. E. 1962. Variations in Progesterone Content of the Bovine Corpus Luteum of the Estrual Cycle. J. Aninlal Sci., 21 : 266. MASON, N. R., AND SAVARD,K. 1964. Specificity of Gonadotropin Stimulation of Progesterone Synthesis in Bovine Corpus Luteum in Vitro. Endocrinology, 74: 664. MAYEI%~D. T., GLASGOW, B. R., AND GAWIENOWSKI, A. 1961. Metabolism of Progesterone and Their Physiological Significance in the ~/rine of Pregnant and NonPregnant Sows and Gilts. J. Animal Sci., 20 : 66. MELA/~(PY,R. M., EIKMERSON, M. A., RAKES, J. M., HANKA, L. J., AND ENESS, P. G. 1957. The Effect of Progesterone on the Estrous Response of Estrogen-Conditioned Ovariectomized Cows. J. Animal Sci., 16: 967. MELAIV~PY,R. M., HEAKN, W. R., AND RAKES, J. M. ]959. Progesterone Content of Bovine Reproductive Organs and Blood During Pregnancy. J. Animal Sei., 18: 307. MIKHAIL, J., NOALL, M. W., AND ALLEN, W. M. 1961. Progesterone Levels in the

(97)

(98)

(99)

(100) (101)

(102)

(103)

(104)

(105)

(106)

(107)

(108)

(109)

(110)

Rabbit Ovarian Vein Blood Throughout Pregnancy. Endocrinology, 69 : 504. MILLER~ W. R., AND TURNER, C. W. 1961. Evidence for an in Vivo Conversion of C21 Steroids to C19 Androgens in the Bovine. J. Dairy Sei., 44: 2278. MILLER, W. R., AND TURNER, C. W. 1963. Biosynthesis of Steroids from Pregnenolone16-H ~ by Bovine Ovarian Tissue in Vitro. Steroids, 2: 657. MILLER, W. R., WILLIAMS, R., PIPES, G. W., AND TURNER, C. W. 1963. Conjugation, Distribution, and Biological Half-Life (t 1~) of Radioactive Progesterone in Plasma and Red Cells of Bovine Blood. J. Dairy Sei., 46: 1402. MIYAKE, T. 1962. Progestational Substances. I n R. I. Dorfman, Ed. Methods in Hormone Research. Vol. II. Academic Press, New York. MOORE, N. W., ROWSON, L. E. A., AND SHORW, R. V. 1960. Egg Transfer in Sheep. Factors Affecting the Survival and Development of Transferred Eggs. J. Reprod. and Fertil., 1: 332. MORRIS, C. J. O. R., AND MORRIS, P. ]963. Separation Methods in Biochemistry. Interscience, New York. NATELSON, S. 1962. Progress in Biochemical Investigations. I. Steroids : 1961. Microchem. J., 6: 381. NEHER, R. 1963. Chromatographic Mobilities. I n L. L. Enge], Ed. Physical Properties of the Steroid Hormones. MacMillan Co., New York. NEUSTAEDTER, P. J. 1963. Selective Oxidations of Polyhydroxyl Steroids and NonCatalytic Selective Reductions of Polycarbonyl Steriods. I n C. Djerassi, Ed. Steroid Reactions. Holden-Day, Inc., San Francisco. NOTEBOOM, W. D., AND GORSKI, J. 1963. An Early Effect of Estrogen on Protein Synthesis. Prec. Natl. Acad. Sci., 50: 250. OERTEL~ G. W., WEISS, S. P., AND EIK-NES~ K . B . 1959. Determination of Progesterone in Human Blood Plasma. J. Clin. Endocrinol. Metabolism, 19: 213. OVF~EE~K, G. A., AND DE VISSER, J. 1964. Different Modes of Action of Two Antiovulatory Compounds. Aeta Endocrinol. Suppl., 90: 179. PATTI, A. A., AND STEIN, A. A. 1964. Steroid Analysis by Gas Liquid Chromatography. C. C Thomas, Springfield, Illinois. PEARL]~IAN, W. H. 1960. Progesterone. In H. N. Antonaides, Ed. Hormones in Human Plasma. Little, Brown and Co., New York. RAESlDE, J. I., AND TURNER, C. W. 1955. Chemical Estimation of Proges*erone in the Blood of Cattle, Sheep, and Goats. J. Dairy Sci., 38: 1334. RICE, B. F., HAIV/MERSTEIN, J., AND SAVARD, K. 1964. Steroid Hormone Formation in

PROGESTERONE IN BOVINE REPRODUCTION

(111)

(112)

(113)

(114)

(115)

(116) (117)

(118)

(119) (120) (121) (122) (123)

(124)

(125)

the Human Ovary. II. Action of Gonadotropins in Vitro in the Corpus Luteum. J. Clin. Endocrinol. Metabolism, 24: 606. RIONDEL, A. M., TAIT, J. F., TAIT, S. A. S., LITTLE, B., AND GUT, M. 1962. The Deterruination of Progesterone and Testosterone in Peripheral Blood with S~-Thiosemicarbazide. (Abstract.) Proc. Ann. Meet. Endocrine Soc. ROMANOFP, E. B., DESHPANDE, N., AND PINCUS, G. 1962. Rate of Ovarian Progesterone Secretion in the Dog. Endocrinology, 70: 532. ROWLANDS, I. W., AND SHORT, R. V. 1959. The Progesterone Content of the Guinea Fig Corpus Lutemn During the Reproducrive Cycle and After IIysterectomy. J. Endocrinol., 19: 18. SOHWEPPE, J. S. 1961. A ]Review of Steroid Hormones. I. Basle Physiological Steroid Concepts with Comments upon Measurement and Fraetionation Techniques. Northwestern Medical School, Evanston, Ill., Quart. Bull., 35 : 248. SEASE, J. W. 1947. The Use of a Fluorescent Adsorbent for the Chromatography of Colorless Compounds. J. Am. Chem. Soe., 69 : 2242. SNORT, R. V. 1956. Progesterone in the Placentae of Domestic Animals. Nature, ] 78 : 743. SHORT, R. V. 1957. Progesterone and Related Steroids in the Blood of Domestic Animals. Colloq. on Endocrinol., CIBA Found., 11: 362. SNORT, R. V. 1958. Progesterone in Blood. I. The Chemical Determination of Progesterone in Peripheral Blood. J. Endocrinol., 16:415. SHOR% R. V. 1958. Progesterone in Blood. II. Progesterone in the Peripheral Blood of Pregnant Cows. J. Endocrinol., 16: 426. SHOR% R. V. 1960. Blood Progesterone Levels in Relation to Parturition. J. Reprod. Fertil., 1: 61. SNORT, R. V. 1961. Progesterone. I n C. H. Gray and A. L. Bacharach, Eds. IIormones in Blood. Academic Press, New York. SNORT, R. V. 1964. Ovarian Steroid Synthesis and Secretion in Vivo. Recent Prog. HHormone Research, 20: 303. SNORT, R. V., AND LEVITT, I. 1962. The Fluerometric Determination of Progesterone in Human Plasma During Pregnancy and the Menstrual Cycle. J. Endocrinol., 25 : 239. SHORT, R. V., McDONALD, M. F., AND ROWSON, L. E. A. 1963. Steroids in the Ovarian Venous Blood of Ewes Before and After Gonadotropic Stimulation. J. Endocrinel., 26 : 155. SNORT, R. V., AND MOORE, N. W. 1959. Progesterone in Blood. V. Progesterone and 20 a-iiydroxypregn-4-ene-3-one in the

(126)

(127)

(128)

(129)

(130)

(131)

(132)

(133)

(134)

(135)

(136)

(137) (138)

(139)

(140)

329

Placenta and Blood of Ewes. J. Endocrinol., 19: 268. SttORT, R. V., AND ROWELL, J. G. 1962. The HHalf-Life of Progesterone in the Peripheral Blood of a Ewe at Two Stages of Gestation. J. Endocrinol., 25: 369. SILBIGER, M., AND ROTHCHILD, I. 1963. The Influence of the Uterus on the Corpus Luteum-PJtuitary Relationship ill the Ra~. Acta Endocrinol., 43: 521. SIIVIMER, H. H., HILLIARD, ,I., AND ARCHIBALg, D. 1963. Isolation and Identification of Progesterone and 20 a-iiydroxypregn-4en-3-one in Ovarian Venous Blood of Rabbits. Endocrinology, 72: 67. SIM1VIONS, K. R., AND HANSEL, W. 1964. Nature of the Luteotrophic Hormone in the Bovine. J. Animal Sci,, 23:136. SO1VIMERVILLE, I. F., PICKETT, M. T., COLLINS, W. P., AND DENYER, D. C. 1963. A Modified Method for the Quantitative Determination of Progesterone in Human Plasma. Acta Endocrinol., 43: 101. SPIES, H. G., ZIlYII~ERI~£AN, D. R., SELF, If. L., AND CASIDA, L. E. 1960. Effect of Exogenous Progesterone on the Corpora Lurer of Hysterectomized Gilts. J. Animal Sci., 19: 101. STAPLES, R. E., AND IIANSEL, "~V. 1961. Luteal Function and Embryo Survival in the Bovine. J. Dairy Sci., 44" 2040. STAPLES, R. E., McENTEE, K., AND RANSEL, W. 1961. Luteal Function as Related to Pituitary and Ovarian Cytology and Embryo Development in the Bovine. J. Dairy Sci., 44: 2049. STORIV[SHAK~ F., AND EI~B, R. E. 1961. Progestins in Bovine Corpora Lutea, Ovaries, and Adrenals During Pregnancy. J. Dairy Sci., 44: 310. STOK~SIIAK, F., INSKEEP, E. K., LYRIC, J. E., POPE, A. L., AN]) CASIDA, L. E. 1963. Progesterone Levels in Corpora Lutea and Ovarian Effluent Blood of the Ewe. J. Animal Sci., 22: 1021. SWEAT, M. L., BERLINEI~, D. L., BEYSON, M. J., NABORS, C., JR., HASKELL, J., AND I-IOLMSTRO~, E. G. 1960. The Synthesis and Metabolism of Progesterone in the Ituman and Bovine Ovary. Biochim. et Biophys. Acta, 40: 289. TAI~IAOKI, B., AND PINCUS, G. 1961. Biogenesis of Progesterone in Ovarian Tissues. Endocrinology, 69: 527. TELEGDY, J. 1963. Die Veranderungen des Progeteron-und A'-Pregnenol-(20 a)-on-(3)Gehalts in Ovargewebe von Ratten wahrend des Ostruszyklus. Endokrinologie, 44: 29. TELEGDY, J. 1963. The Ovarian Secretion of Progesterone and 20 a-Hydroxypregn-4en-3-one in Rats During the Estrous Cycle. Steroids, 2. 119. TEPPEI~I~AI~, J. 1962. Metabolic and En-

330

W. IK. GOMES AND R. E. Et~B

docrine Physiology. Year Book Medical Publishers, Chicago. (141) TEPPERI~£AN~ J., AND TI~PPEI~I~AN~ I~. M. 1960. Some Effects of Hormones on Cells and Cell Constituents. Pharmacol. Revs., 12 : 301. (14~) TO~'H, F. 1964. Effect of Synthetic Progestogens on the Ovaries. Intern. J. l%rti]., 9 : 151. (143) TOUCHSTONE, J. C., AND MUI~AWEC,T. 1960. Enhancement of the Fluorescence of Progesterone (and Other Steroids) in Sulfuric Acid. Anal. Chem., 32: 822. (144) TI~IMBEI~GEI~ G. W., AND HANSEL, W. 1955. Conception Rate and Ovarian Function Following Estrus Control by Progesterone Injection in Dairy Cattle. J. Animal Sei., 14: 244. (145) WIEST, W. G. 1959. Progesterone and 4Pregnen-20 a-ol-3-one in the Tissues of Pregnant Rats. Endocrinology, 65:825. (146) WIEST, W. G., AND FORBES, T. R. 1964. Failure of 20 a-Hydroxy-A~-Pregnen-3-one and 20 fl-Hydroxy-A4-Pregnen-3-oneto Mainrain Pregnancy in Ovariectomized Mice. Endocrinology, 74: 149. (147) WILCOX, R. B., AND WIEST, W. G. 1960. Comparative Effectiveness of 4-Pregnen-20 a-ol-3-one in the Development of Deciduomata. Endocrinology, 67: 281. (14s) WILLIAMS, W. F. 1962. Excretion of Progesterone and Its Metabolites in Milk, Urine, and Feces. J. Dairy Sci., 45: 1541. (149) WILSON, IX., BOI~RIS, ~. J., AND GARRISON, M. M. 1958. Chromatographic Procedure for the Determination of Urinary Corticosteroids and Clo Steroids. J. Clin. Endocrinol. Metabolism, 18: 643. (150) WOOLEVER, C. A. 1963. Daily Plasma Progesterone Levels During the Menstrual Cycle. Am. Y. Obstet. Gyneco]., 85: 981. (~51) WOOLE%rER, C. A., AND GOLDFEII~, A. 1963. A Double-Isotope Derivative Method for Plasma Progesterone Assay. Intern. J. Appl. Radiation and Isotopes, 14: 163.

(152) YANNON:E, M. E., McCoMAS, D. B., AND GOLDFEIN, A. 1964. The Assay of Plasma Progesterone. J. Gas Chromatog., 2: 30. (153) ZAFFARONI,A. 1953. Micromethods for the Analysis of Adrenocortical Steroids. Recent Prog. Hormone Research, 8: 51. (154) gANDER, J. 1962. Progesterone. I n R. I. Dorfman, Ed. Meihods in Hormone Research. Vol. I. Academic Press, New York. (155) ZANDER, J., FORBES, T. R., V0N MUNSTEI~I~ANN, A. M., AND NEHER, R. 1958. A~-3Ketopregnene-20 a-ol and n~-3-Xetopregnene-20 fl-o], Two Naturally Occurring Metabolites of Progesterone. Isolation, Identification, Biologic Activity and Concentration in Human Tissues. J. Clin. Endocrinol. Metabolism, 18: 337. (156) Z~mROW, M. X., NEHER, G. M., LASOWASEM, E. A., AND SALHANICK, H. A. 1957. Biological Activity of Certain ProgesteroneLike Compounds as Determined by the Hooker-Forbes Bioassay. J. C]in. Endocrinol. Metabolism, 17: 658. (157) ZAI~ROW, M. X., YOCHI~i, J. M., AND MCCARTHY, J. L. 1964. Experimental Endocrinology. Academic Press, New York. (158) ZI~BEL~AN, R. G., LOY, ]:~. G., AND CASIDA~ L. E. 1959. Biochemical Studies of tile Bovine Corpus Luteum of Early Pregnancy. J. Animal Sci., 18: 1551. (159) ZIIM[BELMAN, n. G., LOY, R. G., AND CASIDA, L . E . 1961. Variation in Some Biochemical and Histological Characteristics of Bovine Corpora Lutea During Early Pregnancy. J. Animal Sci., 20: 99. (160) ZI~[REL1ViAN, R. G., LOY, R. G., AND CASIDA, L . E . 1961. Effect of Exogenous Progesterone on the Bovine Corpus Luteum of Early Pregnancy. J. Animal Sci., 20:106. (161) ZIMBELMAN, R. G., MCSHAN, W. H., TYLER, W. J., AND CASIDA, L. E. 1961. Effect of a Pituitary Extract on the Bovine Corpus Luteum of Late Pregnancy. J. Animal Sei., 20 : 246.